Toxic organic cations can damage nigrostriatal dopaminergic pathways as seen in most parkinsonian syndromes and in some cases of illicit drug exposure. Here, we show that the organic cation transporter 3 (Oct3) is expressed in nondopaminergic cells adjacent to both the soma and terminals of midbrain dopaminergic neurons. We hypothesized that Oct3 contributes to the dopaminergic damage by bidirectionally regulating the local bioavailability of toxic species. Consistent with this view, Oct3 deletion and pharmacological inhibition hampers the release of the toxic organic cation 1-methyl-4-phenylpyridinium from astrocytes and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration in mice. Furthermore, Oct3 deletion impairs the removal of the excess extracellular dopamine induced by methamphetamine and enhances striatal dopaminergic terminal damage caused by this psychostimulant. These results may have far-reaching implications for our understanding of the mechanism of cell death in a wide range of neurodegenerative diseases and may open new avenues for neuroprotective intervention.astrocytes ͉ Parkinson's disease ͉ extraneuronal monoamine transporter ͉ dopamine ͉ methamphetamine

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148 -7154), we showed that overlapping peptides, N20K (Asn 564 -Lys 583 ) and E20K (Glu 574 -Lys 593 ), from the catalytic domain of phospholipase C (PLC) ␤2 block G␤␥-dependent activation of PLC ␤2. The peptides could also be directly cross-linked to ␤␥ subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to G␤ 1 was inhibited by PLC ␤2 but not by ␣ i1 (GDP), indicating that the peptide-binding site on ␤ 1 represents a binding site for PLC ␤2 that does not overlap with the ␣ i1 -binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC ␤2 interaction with ␤ subunits. Each of the 14 cysteine residues in ␤ 1 were altered to alanine. The ability of the PLC ␤2-derived peptide to cross-link to each ␤␥ mutant was then analyzed to identify the reactive sulfhydryl moiety on the ␤ subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of ␤ 1 which is located in the amino-terminal coiled-coil region of ␤ 1 and identifies a PLC-binding site distinct from the ␣ subunit interaction site.Guanine nucleotide-binding proteins (G proteins) 1 are a large group of structurally similar proteins consisting of three subunits (␣, ␤, and ␥) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the receptor which catalyzes the release of GDP from the ␣ subunit in exchange for GTP (1, 2). In the GDP-bound heterotrimeric state, ␣(GDP)⅐␤␥, neither ␣(GDP) nor ␤␥ can regulate effector activity. Upon receptor-catalyzed G protein activation, the heterotrimer dissociates into free ␣(GTP) and free ␤␥ subunits. It is well understood that both ␣(GTP) and ␤␥ subunits can interact with a variety of downstream effector molecules including enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation of ␣(GDP) with ␤␥ results in deactivation of ␤␥-dependent signaling. Despite detailed knowledge of ␣-and ␤␥ subunit functions, the mechanism for how ␤␥ subunits activate its variety of effectors is not entirely understood.Effector-binding sites on the surface of ␤␥ are beginning to be mapped. The putative competition between ␣(GDP) and effectors for ␤␥ forms the premise for recent studies to map effectorbinding sites at the ␣ subunit-binding interface on ␤. The three-dimensional structure of the G protein heterotrimer reveals that the ␤ subunit is a ␤-propeller with seven "blades" and an amino-terminal ␣-helix (3, 4). The ␣ subunit binds to a portion of the top of the ␤-propeller and along side one of the blades of the propeller. Two groups have shown that alanine substitution of ␣-contacting residues on the top surface...

No abstract